Stephen A. Hitchcock

Structural modifications that alter the P-glycoprotein efflux properties of compounds.

Retrospective analysis of marketed drugs has revealed that very low prevalence of P-glycoprotein (P-gp) efflux and moderate to high passive permeability are two key properties that distinguish drugs which engage targets in the central nervous system (CNS) from those that act predominantly in the periphery. Quantification of the efflux properties that differentiate CNS drugs has been determined using in vitro assays for human and rodent P-gp, and revealed in vivo by comparing brain drug exposures in P-gp (mdr1a/b(−/−)) knockout mice against those observed in wild type mice. Recognition by P-gp not only dramatically affects the distribution properties of compounds into bodily compartments including the CNS, testis, and placenta but also confers resistance to certain cancer chemotherapeutics. This perspective is focused on structural modifications and strategies that can be applied during compound optimization in order to modulate the P-gp efflux properties of small molecules with a particular emphasis on implications for CNS penetration. The topic was last reviewed in 2006 by Raub who noted at the time a surprising paucity of published examples where P-gp efflux had been purposefully circumvented. Greater awareness of the implications of efflux coupled with advances in the quality and throughput of in vitro permeability and efflux assays, greater access to genetic mouse models that lack expression of P-gp, and the development of tool compounds that inhibit efflux transporters has led to an improved understanding of how structural properties influence efflux recognition. However, the precise molecular interactions that confer P-gp efflux remain relatively poorly defined. The promiscuous nature of P-gp renders the rational circumvention of efflux, while simultaneously optimizing compounds for efficacy, safety, and pharmacokinetic properties, an extremely challenging undertaking. The sequence of the human genome has revealed that 500− 1200 genes encode for transport proteins. Energy-dependent active transport proteins are utilized to varying degrees by all cells to traffic a wide variety of substrates including ions, small molecules, and macromolecules against concentration gradients spanning extraand intracellular biological membranes. Active uptake proteins control the translocation of molecules required for healthy cellular function, whereas efflux transporters serve to expel endogenous and exogenous molecules that are recognized as being potentially harmful. Eukaryotic transmembrane transport proteins have been categorized into several subclasses, the two largest being the solute carrier class (SLC) with 348 members identified, and the ATP-binding cassette (ABC) family containing 7 subfamilies (A−G) and 48 identified members. Three ABC proteins; P-gp (encoded by ABCB1 (MDR1)), multidrug resistance protein 1 (MRP1 encoded by ABCC1), and breast cancer resistance protein (BCRP encoded by ABCG2) confer resistance to tumor cells and consequently are among the most extensively studied of the ABC family members. In addition to their role in cancer chemotherapy resistance, P-gp, MRPs, and BCRP also have a significant influence on the absorption, clearance, and penetration of a vast array of small molecules into specific cellular and tissue compartments. P-gp was the first human ABC transporter to be cloned and has been the most extensively studied. P-gp is a glycosylated membrane protein of 170 kDa, and is broadly expressed but particularly enriched in the intestines, liver, kidneys, and at the blood−brain barrier (BBB) and blood−cerebrospinal fluid barrier (BCSFB). P-gp is a highly permissive transporter, recognizing and effluxing a vast diversity of small molecules and peptides. At least 15 additional efflux transporters including members from the organic anion transporter (OAT), multidrug resistance-associated protein (MRP), multidrug resistance protein (MDR), and organic anion transporting polypeptide (OATP) families have been detected at the BBB at the mRNA level. However, P-gp has assumed prominence primarily due to the fact that it has been confirmed experimentally as possessing the broadest substrate specificity.

Targeting cannabinoid agonists for inflammatory and neuropathic pain

The cannabinoid receptors CB1 and CB2 are class A G-protein-coupled receptors. It is well known that cannabinoid receptor agonists produce relief of pain in a variety of animal models by interacting with cannabinoid receptors. CB1 receptors are located centrally and peripherally, whereas CB2 receptors are expressed primarily on immune cells and tissues. A large body of preclinical data supports the hypothesis that either CB2-selective agonists or CB1 agonists acting at peripheral sites, or with limited CNS exposure, will inhibit pain and neuroinflammation without side effects within the CNS. There has been a growing interest in developing cannabinoid agonists. Many new cannabinoid ligands have been synthesized and studied covering a wide variety of novel structural scaffolds. This review focuses on the present development of cannabinoid agonists with an emphasis on selective CB2 agonists and peripherally restricted CB1 or CB1/CB2 dual agonists for treatment of inflammatory and neuropathic pain.

Blood-brain barrier permeability considerations for CNS-targeted compound library design.

A further refinement of the concept of drug-likeness is required for compound libraries intended for central nervous system (CNS) targets to account for the limitations imposed by blood-brain barrier permeability. This review describes criteria and processes that can be applied in the de novo design and assembly of libraries to increase the odds of compounds residing within CNS-accessible chemical space. A number of published examples where CNS activity and/or penetration characteristics have been a factor in library design are discussed.

Design and preparation of a potent series of hydroxyethylamine containing β-secretase inhibitors that demonstrate robust reduction of central β-amyloid.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of β-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS β-amyloid (Aβ) in Sprague-Dawley rats following oral administration.

Discovery and Optimization of a Novel Series of N-Arylamide Oxadiazoles as Potent, Highly Selective and Orally Bioavailable Cannabinoid Receptor 2 (CB2) Agonists

The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC50 > 10 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.

Discovery of potent, selective, and metabolically stable 4-(pyridin-3-yl)cinnolines as novel phosphodiesterase 10A (PDE10A) inhibitors.

We report the discovery of 6,7-dimethoxy-4-(pyridin-3-yl)cinnolines as novel inhibitors of phosphodiesterase 10A (PDE10A). Systematic examination and analyses of structure-activity-relationships resulted in single digit nM potency against PDE10A. X-ray co-crystal structure revealed the mode of binding in the enzymes catalytic domain and the source of selectivity against other PDEs. High in vivo clearance in rats was addressed with the help of metabolite identification (ID) studies. These findings combined resulted in compound 39, a promising potent inhibitor of PDE10A with good in vivo metabolic stability in rats and efficacy in a rodent behavioral model.

Design and synthesis of potent, orally efficacious hydroxyethylamine derived β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-β peptide (Aβ) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aβ levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

A Potent and Orally Efficacious, Hydroxyethylamine-Based Inhibitor of β-Secretase.

β-Secretase inhibitors are potentially disease-modifying treatments for Alzheimers disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to significantly reduce Aβ40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally efficacious BACE inhibitor.

Structural modifications of N-arylamide oxadiazoles : Identification of N-arylpiperidine oxadiazoles as potent and selective agonists of CB2

Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.